The aim of this research is to understand how stable states of gene expression are maintained during development. Molecular, genetic and biochemical methods will be used to study chromatin proteins that control transcription of homeotic (Hox) genes in Drosophila melanogaster. Hox genes determine fly segment identities and their selective expression is needed throughout development. Segmentation gene products control initial patterns of Hox expression in 2-hour-old embryos, but they decay by about 4 hours. The Polycomb group (PcG) proteins are transcriptional repressors that then maintain Hox expression patterns during the rest of development. Current models suggest repression is maintained through covalent histone modification and stable association of PcG protein complexes in the local chromatin. This research will investigate molecular roles of PcG complexes and their subunits. Two biochemically separable PcG complexes have been purified; the ESC-E(Z) complex contains the PcG proteins Extra sex combs (ESC), Enhancer of zeste [E(Z)] and Suppressor of zeste-12 [SU(Z)12]. This complex has an enzyme activity that methylates histones. The other complex, called PRC1, contains Polycomb (PC), Polyhomeotic (PH), Posterior sex combs (PSC) and Sex comb extra (SCE). This work will address mechanisms that regulate the ESC-E(Z) complex and define roles of individual subunits. Another goal is to determine if and how the Sex comb on midleg (SCM) repressor works with PRCI. Additional studies will track PcG chromatin states during the cell cycle. The methods used will include protein purification, enzyme assays, site-directed mutagenesis, chromatin analyses, loss-of-function studies, transgene manipulating, protein interaction tests, and immunostaining of chromosomes. Every fly PcG repressor has homologs in mammals. These are functional homologs since PcG knockouts produce Hox defects in mouse embryos that parallel defects in fly PcG mutants. Human PcG complexes resemble their fly counterparts in composition and activity. Thus, determination of PcG mechanisms in flies should yield insight about developmental controls in higher organisms. PcG repressors are implicated in lymphomagenesis and in normal hematopoiesis. The human homolog of E(Z) is implicated in disease progression of breast and prostate cancers. Knowledge about PcG mechanisms in flies may thus improve understanding of normal blood cell development and processes that underlie these human cancers.